Reversed phase chromatography (RPC-5) and reversed salt gradient chromatography (Sepharose 4B) have been set up recently. A 75Se-tRNA, separated from Pseudomonas cells, was identified as L-leucyl-tRNA and further separated from its non-selenium containing isoacceptors by RPC-5. A quite pure seleno-tRNA from C. sticklandii was separated from its bulk tRNA mixture after two reversed salt gradient chromatographic steps and two reversed phase chromatographic steps. Preliminary results indicate this seleno-tRNA exhibits an unusual absorbance at 291 nm which may be a contribution of the seleno base present in the preparation.